Existence of functional lingual antimicrobial peptide in bovine milk

Abstract

The lingual antimicrobial peptide (LAP) belongs to the beta-defensin family in cattle and is localized in epithelial cells of alveoli in mammary glands. The aim was to investigate whether LAP is secreted into milk and whether the secreted LAP has antimicrobial activity. Decaseinated bovine skim milk was applied to sample extraction cartridges, and the eluate was used for competitive enzyme immunoassay and Western blotting to test for the presence of LAP in milk. After tricine-SDS PAGE, the gel was stained using the periodic acid-Schiff reaction to examine the possibility of glycosylation of LAP. The eluate obtained from the sample extraction cartridges was subjected to a LAP antibody-coupled affinity column, after which the antimicrobial activity of its eluate against Escherichia coli was investigated with radial diffusion plate assay and colony-forming unit enumeration following the culture of bacteria with the sample. The immunoreactive LAP was detected in the eluate by competitive enzyme immunoassay (optical density = 0.437 +/- 0.012 vs. 0.468 +/- 0.016). In the Western blotting analysis, immunoreactive bands were seen around 8, 14, and 17 kDa. The bands at 14 and 17 kDa, but not 8 kDa, were periodic acid-Schiff reaction-positive. The eluate of LAP antibody-coupled affinity column had antimicrobial activity against E. coli (cfu/control = 0.17 +/- 0.18). These results suggest that bovine milk contains functional LAP-like substances that exert antimicrobial activity.